This invention concerns fused azepinone cyclin dependent kinase inhibitors, compositions comprising these compounds, and methods for administering such compounds for diseases of cellular proliferation and/or abnormal protein phosphorylation.
A major advance in the understanding of the control of the cell cycle has been the discovery of a family of enzymes called cyclin dependent kinases (cdk). Structurally they consist of a catalytic subunit and a regulatory subunit. The catalytic subunit is similar to the catalytic region in a number of serine/threonine kinases and at least eight distinct subunits have been described (cdk1(=cdc2)-cdk8). The regulatory subunit is necessary for activity and a number of proteins in this family have been described (cyclin A-cyclin H). Most cyclins can interact with more than one cdk and each known cyclin-cdk pair seems to have a distinct role in regulating the cell cycle. These activities are regulated not only through transcriptional and translational control of the subunits, but also through phosphorylation and dephosphorylation of the subunits. In addition, negative regulatory proteins have been discovered (p15, p16INK4, p21cip1 and p27Kip1) which bind to the cyclin-cdk complex and inhibit activity. Structural understanding of the cdks and their regulation has been advanced by the solution of crystal structures for cdk2, cyclin A, cdk2-cyclin A, and cdk2-cyclin A-p27Kip1. [Russo et al., xe2x80x9cCrystal Structure of the P27Kip1 Cyclin-Dependent Kinase-4 Bound to the Cyclin-A-Cdk2 Complex,xe2x80x9d Nature, 382(6589):325-331 (1996).]
It is clear then that the cdks are important in the control of the cell cycle. As a result, it appears that alterations in cdk expression, function or regulation are associated with diseases of cellular proliferation. Alterations that would increase cdk activity (overexpression of the catalytic and/or positive regulatory subunit, or underexpression or deletion of negative regulatory proteins) have been reported in many cancers. The most common observation has been the deletion of the pl6 (also called MTS1, CDKN2, p16INK) gene. This gene codes for a protein that inhibits the activity of cdk4 and cdk6. This loss of inhibitory activity has been observed in a wide variety of primary human tumors and human tumor-derived cell lines, including lung, breast, brain, bone, skin, bladder, kidney, ovary, liver, colon, pancreas and leukemias. Overexpression of cdk1 in ovarian carcinoma and overexpression of cyclin D in non-small cell lung cancer also has been observed.
Clinical studies have shown that alterations in cdk pathways have prognostic significance. Deletion of the p 16 gene has shown to be associated with poor prognosis in B cell lymphomas [R. Garcia-Sanz et al., xe2x80x9cDeletions and Rearrangement of Cyclin-Dependent Kinase 4 Inhibitor Gene p16 are Associated with Poor Prognosis in B cell Non-Hodgkin""s Lymphomas,xe2x80x9d Leukemia, 11(11):1915-20 (1997)], and pediatric acute lymphoblastic leukemia [e.g., U. R. Kees et al. xe2x80x9cHomozygous Deletion of the p16/MTS1 Gene in Pediatric Acute Lymphoblastic Leukemia is Associated with Unfavorable Clinical Outcome,xe2x80x9d Blood, 89(11):4161-6 (1997)]. High expression of cyclin D1 has also been shown to predict early relapse in pediatric ALL. [U. R. Kees et al., xe2x80x9cDeletions of the p16 Gene in Pediatric Leukemia and Corresponding Cell Lines,xe2x80x9d Oncogene, 12(10):2235-9 (1996).] High expression of cdk1 predicts disease recurrence in prostate adenocarcinoma. [B. V. Kallakury et al., xe2x80x9cThe Prognostic Significance of p34cdc2 and Cyclin D1 Protein Expression in Prostate Adenocarcinoma,xe2x80x9d Cancer, 80(4):753-63 (1997). Loss of p21 expression resulted in a significantly higher risk of recurrence following surgery for gastric carcinoma. M. Ogawa et al., xe2x80x9cLoss of p21WAF1/CIP1 Expression Correlates with Disease Progression in Gastric Carcinoma,xe2x80x9d Br. J Cancer, 75(11):1617-20 (1997).] Higher p27 expression has correlated with longer survival times in breast [e.g., C. Catzavelos et al., xe2x80x9cDecreased Levels of the Cell-Cycle Inhibitor of p27KIP1 Protein: Prognostic Implications in Primary Breast Cancer,xe2x80x9d Nat. Med., 3(2):227-30 (1997)], and non-small cell lung cancer [V. Esposito, xe2x80x9cPrognostic Role of the Cyclin-Dependent Kinase Inhibitor p27 in Non-Small Lung Cancer,xe2x80x9d Cancer Res., 57(16):3381-5 (1997)].
Atherosclerosis is another disease associated with excessive cellular proliferation. An important signal for proliferation of vascular smooth muscle cells is increased expression of cdk2 and associated regulatory subunits, cyclin E and cyclin A. [E.g., C. Ihling, et al., xe2x80x9cTopographical Association Between the Cyclin-Dependent Kinases Inhibitor P21, p53 Accumulation, and Cellular Proliferation in Human Atherosclerotic Tissue,xe2x80x9d Arterioscler. Thromb. Vasc. Biol., 17(10):2218-24 (1997).] This is consistent with the observation that high levels of homocysteine, known to cause occlusive arterial disease, causes increases in aortic cdk activity. [B. Lubec et al., xe2x80x9cHomocysteine Increases Cyclin-dependent Kinase in Aortic Rat Tissue,xe2x80x9d Circulation, 94(10):2620-5 (1996).] The involvement of cdk2 also is consistent with the report that an antisense cdk2 oligonucleotide can prevent graft coronary arteriosclerosis. [J. Suzuki et al., xe2x80x9cPrevention of Graft Coronary Arteriosclerosis by Antisense cdk2 Kinase Oligonucleotide,xe2x80x9d Nat. Med., 3(8):900-3 (1997).]
Other diseases in which there is evidence that inhibitors of cdks may be of therapeutic use include mesangial proliferative glomerulonephritis [J. W. Pippin et al., xe2x80x9cDirect in vivo Inhibition of the Nuclear Cell Cycle Cascade in Experimental Mesangial Proliferative Glomerulonephritis with Roscovitine, a Novel Cyclin-Dependent Kinase Antagonist,xe2x80x9d J. Clin. Invest., 1900(9):2512-20 (1997)], infection with human cytomegalo-virus [W. A. Bresnahan et al., xe2x80x9cInhibition of Cellular cdk2 Activity Blocks Human Cytomegalovirus Replication,xe2x80x9d Virology, 231(2):239-47 (1997)], and malaria [R. Graeser et al., xe2x80x9cPlasmodium Falciparum Protein Kinase 5 and the Malarial Nuclear Division Cycles,xe2x80x9d Mol. Biochem. Parasitol, 82(1):37-49 (1996)]. Abnormal phosphorylation of tau protein is a characteristic of Alzheimer""s disease. Recent reports have shown that this phosphorylation is carried out, at least in part, by brain cdk5 [e.g., A. Sengupta et al., xe2x80x9cPotentiation of GSK-3-Catalyzed Alzheimer-like Phosphorylation of Human tau by cdk5,xe2x80x9d Mol Cell. Biochem., 167(1-2):99-105 (1997)]. Inhibitors of cdk5 should be useful in the treatment of the disease.
The potential role of cdk inhibitors in therapy of numerous diseases has led to efforts to find small molecules that inhibit all or some of the cdks. Several small molecules have been discovered that inhibit cdks specifically. These include the purine analogs, olomoucine, roscovitine, and CVT-313; the flavonoid, flavopiridol; and butyrolactone I. Other potent inhibitors of cdks are known, including staurosporine, UCN-01, and suramin, but these compounds also are potent inhibitors of other protein kinases. Crystal structures for several specific cdk inhibitors in complex with cdk2 have been determined, including olomoucine, roscovitine, and flavopiridol. One specific inhibitor of cdks has reached clinical trials, flavopiridol [H. H. Sadlacek et al., xe2x80x9cFlavopiridol (186-8275, NSC-649890), a New Kinase Inhibitor for Tumor Therapy,xe2x80x9d International Journal of Oncology, 9:1143 (1996)], which has shown antitumor activity in Phase I trials in a number of tumor types and is progressing to Phase II trials. All other known cdk inhibitors have been shown to inhibit the growth of tumor cells in culture, although none are as potent as flavopiridol. No in vivo anti-tumor studies have been reported for these compounds, although there is an anecdotal report of a response to olomoucine in a spontaneous dog melanoma.
Several cdk inhibitors have shown activity in models for other diseases. Animal studies have shown that CVT-313 is an effective inhibitor of neointimal proliferation in a rat restenosis model. [E. E. Brooks et al., xe2x80x9cCVT-313, a Specific and Potent Inhibitor of CDK2 that Prevents Neointimal Proliferation,xe2x80x9d J. Biol. Chem., 272(46):29207-11 (1997).] Roscovitine has been reported to improve renal function in a rat model of glomerulonephritis, and to be an inhibitor of human cytomegalovirus replication in culture. Roscovitine also inhibits DNA synthesis in plasmodiumfalciparum, the malarial parasite. R. Graser et al., supra. 
7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one is a known compound. This compound originally was synthesized by Dr. Conrad Kunick, then at the Pharmazeutisches Institut der Universitxc3xa4t Bonn. C. Kunick, xe2x80x9cSynthese von 7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-onen und 6,11-dihydro-indolo[3xe2x80x2, 2xe2x80x2: 2,3]azepino[4,5-b]indol-5(4H)-on,xe2x80x9d Arch. Pharm. (Weinheim), 325:297-299 (1992).
From the above discussion it is clear that known cyclin-dependent kinase inhibitors are useful for treating diseases of cellular proliferation and/or abnormal protein phosphorylation. But, there still is a need for new potent, and preferably selective, cdk inhibitors.
The present invention provides a new class of cyclin dependent kinase inhibitors that also have shown antiproliferative activity in human tumor cell line assays. These compounds typically satisfy the formula 
wherein A is oxygen or sulfur coupled to the ring by a single or a double bond, Y and Z are conjugated rings, the Y ring has at least one carbon atom with a substituent R1 selected from the group consisting of alkoxy, amino, acyl, aliphatic substituents, particularly alkyl, alkenyl and alkinyl substituents, and even more particularly lower aliphatic substitutents, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl and imino, R2 is selected from the group consisting of hydrogen, benzyl, lower alkyl, and lower alkyl ester, the Z ring has a substituent R3 selected from the group consisting of hydrogen, lower aliphatic substituents, particularly lower alkyl substituents, or cyclic alkyl. xe2x80x9cLowerxe2x80x9d as used herein typically refers to compounds or substituents having 10 or fewer carbon atoms in a chain, and includes all position, geometric and stereoisomers of such substituents or compounds. Furthermore, if R1 and R2 are hydrogen and the Z ring comprises a five membered ring fused to a six membered ring then the six membered ring includes a substituent other than bromine. A is most typically a double bond, and the majority of compounds made to date have A double bonded to oxygen.
Most of the compounds of the present invention further satisfy the formula wherein A is oxygen or sulfur coupled to the ring by a single or double bond, R2 is selected from the group consisting of hydrogen, aryl, lower aliphatic substituents, particularly alkyl and 
lower alkyl ester, R4-R7 are independently selected from the group consisting of alkoxy, amino, acyl, aliphatic substituents, particularly alkyl, alkenyl and alkinyl substituents, aliphatic alcohols, particularly alkyl alcohols, aliphatic nitrites, particularly alkyl nitrites, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl, imino, and xcex1, xcex2 unsaturated ketones, R8-R1, are independently selected from the group consisting of aliphatic substituents, particularly alkyl, alkenyl and alkinyl substituents, particularly lower aliphatic substituents, aliphatic alcohols, particularly alkyl alcohols, alkoxy, acyl, , cyano, nitro, epoxy, haloalkyl groups, halogen, hydrogen, hydroxyl, cyano groups, and nitro groups, and R12 is selected from the group consisting of aliphatic groups, particularly lower alkyl groups, aliphatic alcohols, particularly alkyl alcohols, carboxylic acids, and hydrogen. Particular examples of compounds satisfying these formulas have: R2 selected from the group consisting of H, xe2x80x94CH2COOCH3, xe2x80x94CH3, and xe2x80x94CH2Ph, preferably hydrogen; R4-R7 independently selected from the group consisting of alkoxy, amino, acyl, alkyl, alkenyl, alkinyl, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl and imino, lower aliphatic alcohols, lower aliphatic nitrites, and xcex1, xcex2 unsaturated ketones, cyano groups, and nitro groups, particularly xe2x80x94H, xe2x80x94OH, xe2x80x94C(xe2x95x90NH)NH2, xe2x80x94CO2H, xe2x80x94Br and xe2x80x94OCH3; R8-R11, independently selected from the group consisting of alcohols, alkoxy, acyl, alkyl, alkenyl, alkinyl, cyano, nitro, epoxy, haloalkyl, halogen, hydrogen, hydroxyl and lower alkyl, particularly xe2x80x94H, halogens, xe2x80x94OH, xe2x80x94CH2OH, xe2x80x94CH2CHOCH2, xe2x80x94CH2CH2CHOCH2, xe2x80x94CF3 and xe2x80x94OCH3; and R12 selected from the group consisting of alcohols, carboxylic acids, hydrogen and lower alkyl groups, particularly xe2x80x94H, xe2x80x94CH2CH2OH, xe2x80x94CH3 and xe2x80x94CH2CH3.
The present invention also provides compositions comprising effective amounts of a compound, or compounds, satisfying the formulas above. Such compositions may further comprise inert carriers, excipients, diagnostics, direct compression binders, buffers, stabilizers, fillers, disintegrants, flavors, colors, lubricants, other active ingredients, other materials conventionally used in the formulation of pharmaceutical compositions, and mixtures thereof.
A method for inhibiting the proliferation of living cells in a subject, such as hyperproliferative cells and/or neoplastic cells, or for treating a neoplasm in a subject also is provided. The method first comprises providing a compound or compounds, or a composition comprising the compound or compounds, as described above. An effective amount of the compound(s) or composition(s) is then administered to a subject to inhibit the proliferation of living cells. Administering the compound(s) or composition(s) generally comprises administering topically, orally, intramuscularly, intranasally, subcutaneously, intraperitoneally, intralesionally or intravenously. The currently preferred administration method is intravenous. The effective amount should be as high as the subject can tolerate, but typically is from about 0.1 gram to about 3.0 grams of a compound, and from about 30 mg/kg of subject/dose to about 400 mg/kg of subject/dose, preferably from about 30 mg/kg of subject/dose to about 50 mg/kg of subject/dose.